DNA origami surpasses important thresholds

Building virus-sized structures and saving costs through mass production

2017-12-07 – News from the Physics Department

It is the double strands of our genes that make them so strong. Using a
technique known as DNA origami, biophysicist Hendrik Dietz has been building
nanometer-scale objects for several years at the Technical University of Munich
(TUM). Now Dietz and his team have not only broken out of the nanometer realm to
build larger objects, but have also cut the production costs a thousand-fold.
These innovations open a whole new frontier for the technology.

Viruses encapsulate their genetic material in a shell comprising a
series of identical protein building blocks. The hepatitis B virus
capsule, for example, comprises 180 identical subunits, a typical case
of “prefabricated” construction deployed frequently in nature.

The team led by Hendrik Dietz, Professor of Biomolecular Nanotechnology
at the TU Munich has now transferred viral construction principles to
DNA origami technology. This allows them to design and build structures
on the scale of viruses and cell organelles.

The technology builds on a long single strand that is appended to a
double-stranded structure using short staple sequences. “The
double-stranded structure is energetically sufficiently stable so that
we can force the single strand into almost any shape using appropriately
chosen counterparts,” explains Hendrik Dietz. “This way we can precisely
design objects in the computer that are merely a few nanometers in
size.”

Gears for nanomotors

“V-shaped” building blocks, constructed using DNA origami techniques, form
„gear-wheels“ by self-organization. The number of elements is determined by the
opening angle of the building block. In a next step, these gears form tubes with
a size comparable with virus capsids.
– Image: Hendrik Dietz / TUM

The Dietz lab commands techniques that allow them to further modify and
insert chemical functionalities into objects by adding side groups. But,
until now, the size of the objects remained in the nanometer realm. In
the renowned scientific journal Nature, the team now describes how
larger structures can be built using prefabricated parts.

To this end, they first created V-shaped nano-objects. These have
shape-complementary binding sites on their sides, allowing them to
autonomously attach to each other while floating in a solution.
Depending on the opening angle, they form “gears” with controlled number
of spokes.

“We were thrilled to observe that, almost without exception, rings
formed as defined by the opening angle,” says Hendrik Dietz. “Decisive
for the ability to build objects of this size and complexity is the
precision and rigidity of the individual building blocks. We had to
reinforce individual elements with crossbars, for instance.”

Construction of microtubes

To further exploit the construction principle, the team created new
building blocks that had “glue joints” not only on the sides, but also
slightly weaker ones on the top and bottom. This allows the “nano-gears”
to form long tubes using the additional docking sites in a second step.

“At lengths of one micrometer and a diameter of several hundred
nanometers, these tubes have reached the size of some bacteria,”
explains Hendrik Dietz. “And we can use the architecture of individual
elements to determine features of the overall structure.”

Building polyhedral structures

Inspired by the symmetries and the hierarchical design of viruses, the
researchers also attempted to build closed cage structures. “A potential
future application of artificial cages is the transport of medication in
the body,” explains Hendrik Dietz. “Here, the goal is to release active
agents only at specific desired locations, sparing the rest of the
body.”

Using the principles already applied to the structures described before,
the team now constructed new elements they hoped would assemble in a
self-limiting fashion into cage structures under the right conditions.
According to these strategies a triangular middle section and three
V-shaped elements give rise to a three-pronged building element.

Depending on the opening angle of the V, a defined number of these units
merge to form tetrahedral, hexahedral or dodecahedral structures in a
second step. The final structures integrate up to 1.8 million
addressable DNA base pairs at user-defined positions. For the first
time, these discrete-size cages attain molecular weights and sizes
comparable to that of viruses and small cell organelles.

Cost-effective mass production

DNA origami object in the form of a screw-nut.
On the side one can see how the long DNA single strand (blue) is forced into
the predefined shape by shorter “staple strands”.
– Image: Hendrik Dietz / TUM

To date, manufacturing processes have limited the scope of application
to those requiring only small amounts of material. The fact that only a
few micrograms can be manufactured with conventional methods precludes
many potential medical and materials science applications.

The bottleneck is the short staple strands that must be chemically
produced base by base. The main strand obtained from bacteriophages, on
the other hand, can be produced on a large scale using biotechnological
processes.

That is why the team led by Hendrik Dietz refined so-called DNA enzymes,
a discovery stemming from synthetic biotechnology. These are DNA strands
that break apart at specific positions when exposed to a high
concentration of zinc ions.

They joined the short staple sequences to a long strand using two
modified DNA enzymes each. “Once precisely assembled with a specific
base sequence, these combined strands can be reproduced in a
biotechnological process, as with single strands of bacteriophage DNA,”
says Dietz, explaining the key feature of the process.

Biotechnological production on a large scale

Both the main strand and the secondary strand, comprising DNA enzymes
and the staple sequences, were successfully produced using a high cell
density process with bacteria. The process is scalable and thus amenable
to high volume production of the main strands and staples. Increasing
the zinc ion concentration after DNA isolation releases the short staple
sequences, which then fold the main strand into the desired shape.

Extensive investigations of the reaction mechanisms in collaboration
with colleagues at the Institute of Biochemical Engineering showed that
this is possible even on a large scale. At the TUM Research Center for
Industrial Biotechnology in Garching, scientists have now produced
multiple grams of four different DNA origami objects. Scaling up the
process to a cubic meter scale is now within grasp.

“The interplay of biotechnology and process technology has thus enabled
setting a truly fundamental milestone on the path to future applications
in DNA nanotechnology,” says Professor Dirk Weuster-Botz, Chair of the
Institute of Biochemical Engineering.

The research was funded by the European Research Council, the German
Research Foundation (DFG) through the Gottfried-Wilhelm-Leibniz Program,
the collaborative research project SFB 863, the Excellence Cluster for
Integrated Protein Science Munich (CIPSM) and Nanosystems Initiative
Munich (NIM), the International Graduate School of Science and
Engineering, the Institute for Advanced Study of the Technical
University of Munich using funds from the DFG and the European
Community, the BioOrigami project of the German Federal Ministry of
Education and Research (BMBF), and the Bosch Research Foundation.